Glutathione (GSH) is a ubiquitous and vital tripeptide in mammalian organisms. It plays a critical role in detoxification of xenobiotics, electrophiles, peroxides and oxygen radicals. About 5% of cellular oxidative metabolism produces radical forms of oxygen. It has been proposed that the cumulative, irreversible damange by this process to cellular structure/function could be a key mechanism of aging. since GSH is the major intracellular defense agent against this process, its homeostasis and regulation in the aging organism is of utmost importance. The key role of the liver is hepatic and extrahepatic GSH metabolism makes it the organ of prime importance in GSH metabolism. The overall purpose of this proposal is to delineate the characteristics and mechanisms of changes in turnover and efflux of hepatic GSH as a function of age. In particular the following specific aims will be pursued: (1) Identification of changes inthe kinetics of hepatic sinusoidal and canalicular GSH efflux as a function of age. We will define whether the observed decline in the carrier-mediated sinusoidal efflux with age is due to changes in Vmax; Km, or both. We will also accurately define the changes in the kinetics of biliary efflux with age, by blocking biliary hydrolysis of GSH using AT-125. (2) Determination of the mechanisms of changes in the heptatic GSH turnover with age. Using improved designs and methods, the turnover rate of hepatic GSH turnover rate of hepatic GSH and its relationship to sinusoidal and biliary efflux will be measured by tracer-kinetic methods and multicompartmental analysis. (3) Delineation of the effect of perturbation of hepatic GSH pool size on turnover and efflux. These studies will probe in more depth the relationships and quantitative contribution of sinusoidal and canalicular efflux to the total hepatic turnover and provide more rigorous testing of the homogeneity of the hepatic GSH pool. (4) Determination of the role of availability of sulfur amino acids. These studies will explore the age-related correlates of hepatic uptake of methionine and cysteine. (5) Development of kinetic, compartmental model of hepatic GSH turnover and efflux in aging. The results of our experiments will be analyzed by multicompartmental methods with the aim of integrating the findings into a comprehensive model of GSH turnover and efflux in aging.